Enzymes and methods for dealkylation of substrates

ABSTRACT

Disclosed herein are enzymes and organisms useful for the dealkylation of products derived from lignin depolymerization, including the conversion of guaiacol or guaethol to catechol or the conversion of anisole to phenol. Methods of converting guaiacol or guaethol to catechol or anisole to phenol using enzymes or organisms expressing the same are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/130,482, filed Mar. 9, 2015, the contents of which are incorporatedby reference in their entirety.

CONTRACTUAL ORIGIN

The United States Government has rights in this invention under ContractNo. DE-AC36-08GO28308 between the United States Department of Energy andAlliance for Sustainable Energy, LLC, the Manager and Operator of theNational Renewable Energy Laboratory.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file entitled “14-54_ST25.txt,” having a size in bytes of 56 kb andcreated on Mar. 8, 2016. Pursuant to 37 CFR §1.52(e)(5), the informationcontained in the above electronic file is hereby incorporated byreference in its entirety.

BACKGROUND

Lignocellulosic biomass represents a vast resource for the production ofrenewable transportation fuels and chemicals to offset and replacecurrent fossil fuel usage. The lignin component of lignocellulosicbiomass is an energy-dense, heterogeneous alkyl-aromatic polymercomprised of phenylpropanoid monomers used by plants for water transportand defense, and it is the second most abundant biopolymer on Earthafter cellulose. Lignin is typically underutilized in most selectiveconversion processes for biofuel production. In the production of fuelsand chemicals from biomass, lignin is typically burned for process heatbecause its inherent heterogeneity and recalcitrance make it difficultto selectively upgrade the monomers to value added products. Thislimited ability to utilize lignin, despite being the most energy densepolymer in the plant cell wall, is primarily due to its inherentheterogeneity and recalcitrance. Guaiacol (2-methoxyphenol) is one ofmany products that result from lignin depolymerization.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods that aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

Exemplary embodiments provide methods for removing an alkyl group froman aromatic substrate by contacting material containing the aromaticsubstrate with a cytochrome P450 polypeptide and a reductase polypeptideto generate a dealkylation product.

In various embodiments, the contacting step comprises culturing amicroorganism with the material containing the aromatic substrate wherethe microorganism expresses an exogenous gene encoding a cytochrome P450polypeptide or a reductase polypeptide.

In some embodiments, the aromatic substrate comprises guaiacol, anisoleor guaethol; comprises products of lignin depolymerization, or comprisesa pyrolysis oil or bio-oil.

In certain embodiments, at least one of the cytochrome P450 polypeptideor the reductase polypeptide is from a bacterium, such as a bacteriumfrom the genera Amycolatopsis or Rhodococcus.

In exemplary embodiments, the cytochrome P450 polypeptide has an aminoacid sequence at least 90% identical to SEQ ID NO:2. In others, thereductase polypeptide has an amino acid sequence at least 90% identicalto SEQ ID NO:4.

In further embodiments, the dealkylation product is catechol or phenol.

In some embodiments, the methods further comprise isolating thedealkylation product.

Additional embodiments provide isolated cDNA molecules encodingcytochrome P450 polypeptides that have amino acid sequences at least 90%identical to SEQ ID NO:2 or encoding reductase polypeptides that haveamino acid sequences at least 90% identical to SEQ ID NO:4. In certainembodiments, the polypeptides have amino acid sequences identical to SEQID NO:2 or SEQ ID NO:4.

In various embodiments, the cDNA molecules further comprise an exogenouspromoter operably linked to the cDNA molecules. Other embodimentsprovide expression vector comprising the cDNA molecules and host cellsthat express a recombinant polypeptide encoded by the cDNA molecules,including host cells that are strains of Pseudomonas such as P. putida.

Other embodiments provide isolated cytochrome P450 polypeptides andreductase polypeptides encoded by the cDNA molecules.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than limiting.

FIG. 1 shows exemplary dealkylation reactions catalyzed by the twocomponent cytochrome P450 system.

FIG. 2 shows the nucleic acid sequence (A) and amino acid sequence (B)of a cytochrome P450 O-dealkylase from Amycolatopsis sp. ATCC 39116.

FIG. 3 shows the nucleic acid sequence (A) and amino acid sequence (B)of a reductase from Amycolatopsis sp. ATCC 39116.

FIG. 4 shows growth by P. putida KT2440 engineered to express acytochrome P450 O-dealkylase and a reductase (P. putida KT2440/pCJ021)on media containing guaiacol.

FIG. 5 shows guaiacol and catechol levels for P. putida KT2440 and P.putida KT2440/pCJ021 after growth on media containing guaiacol.

FIG. 6 shows guaiacol levels for P. putida KT2440 and P. putidaKT2440/pCJ021 after growth on media containing guaiacol.

FIG. 7 shows phenol levels for P. putida KT2440 and P. putidaKT2440/pCJ021 after growth on media containing anisole.

FIG. 8 shows guaethol levels for P. putida KT2440 and P. putidaKT2440/pCJ021 after growth on media containing guaethol.

FIG. 9 shows growth of P. putida KT2440 engineered to express acytochrome P450 O-dealkylase and a reductase (P. putida KT2440/pCJ021)on media containing guaethol.

DETAILED DESCRIPTION

Disclosed herein are enzymes, organisms expressing these enzymes, andmethods useful for the dealkylation of aromatic substrates, includingthe conversion of guaiacol or guaethol to catechol and the conversion ofanisole to phenol. Methods of converting aromatic substrates found inlignin-based feedstocks such as pyrolysis oil into catechol and phenolare also disclosed. FIGS. 2 and 3 present the nucleic acid sequences andamino acid sequences of two enzymes useful for the enzymatic conversiondescribed herein.

Guaiacol (2-methoxyphenol), anisole (methoxybenzene), and guaethol(2-ethoxyphenol) are common products of lignin depolymerization, and theconversion of guaiacol or guaethol to catechol (1,2-dihydroxybenzene)allows the more efficient use of products derived from lignin.

Disclosed herein are cytochrome P450 O-dealkylases and reductases thatcatalyze reactions such as the O-demethylation of guaiacol to catechol,the O-demethylation of anisole to phenol, and the O-deethylation ofguaethol to catechol. Generally, dealkylation is the removal of an alkylgroup from a substrate, such as the removal of a methyl group to convertguaiacol to catechol and formaldehyde. These enzymes have activity notonly on guaiacol, but also on anisole and guaethol. O-dealkylases mayremove methyl, ethyl, propyl, butyl and other alkyl groups of thegeneral formula C_(n)H_(2n+1) from substrates. Another O-dealkylaseactivity may be to perform ether bond cleavage on aromatic compounds. Invarious embodiments, the enzymes may be from the CYP255 family ofcytochrome P450 enzymes.

Some bacterial cytochrome P450 enzymes cooperate with one or two partnerproteins, usually a reductase and a ferredoxin, that transfer electronsfrom a cofactor such as NAD(P)H to the cytochrome, though there arevariations on this throughout prokaryotes and eukaryotes. In certainembodiments, the reductase may comprise a 2Fe-2S ferredoxin domain, aflavin adenine dinucleotide (FAD) binding region, a nicotinamide adeninedinucleotide (NAD) binding region or combinations thereof. For example,the reductase represented by SEQ ID NO:4 comprises an N-terminal 2Fe-2Sferredoxin domain followed by a FAD and NAD binding region with homologyto ferredoxin-NADPH reductase (FNR) type oxidoreductases. This domainarchitecture is novel for a cytochrome P450 reductase, and the presenceof both ferredoxin and NAD binding domains may indicate the reductaseand the cytochrome P450, whose genes are natively clustered andtranscribed together, form a two-component cytochrome P450 system.

The combination of both the cytochrome P450 and reductase polypeptidesmay be used as a two-component P450 system for dealkylating aromaticsubstrates, including demethylating guaiacol to produce catechol. Forexample, nucleic acid molecules encoding SEQ ID NOs: 2 and 4 encode atwo-component cytochrome P450 system with guaiacol O-demethylaseactivity, as well as activity on other substrates including anisole(methoxybenzene), guaethol (2-ethoxyphenol), 2-propoxyphenol, and othersubstituted O-alkoxyphenols. Additional examples of two-componentsystems that exhibit these enzymatic activities include the cytochromeP450 (EHK82401; SEQ ID NO:6) and reductase (EHK82400; SEQ ID NO:8)polypeptides from Rhodococcus pyridinivorans AK37 and the cytochromeP450 (WP_011595125; SEQ ID NO:10) and reductase (WP_011595126; SEQ IDNO:12) polypeptides from Rhodococcus jostii.

In addition to the enzymes described in FIGS. 2 and 3 and in theSequence Listing, other suitable enzymes for aromatic substrateconversion include enzymes from Rhodococcus pyridinivorans strainsSB3094 (e.g., YP_008987954.1) and AK37 (e.g., WP_006553158.1),Rhodococcus jostii RHA1 (e.g., YP_702345.1) and Amycolatopsis sp. ATCC39116 (previously known as Streptomyces setonii or Streptomyces griseusstrain 75iv2). In some embodiments, the cytochrome P450 or reductasepolypeptide may be from a species of the genera Rhodococcus (e.g., R.pyridinivorans or R. jostii) or Amycolatopsis (e.g., Amycolatopsis sp.ATCC 39116). Additional exemplary cytochrome P450 and reductasepolypeptides are provided in Table 1 below.

In various embodiments, the cytochrome P450 and reductase polypeptidesmay be from microorganisms such as bacteria, yeast or fungi. Exemplarybacteria include species from the family Pseudonocardiaceae or speciesfrom the genera Rhodococcus, Amycolatopsis, Pimelobacter, Gordonia,Pseudonocardia, Saccharomonospora, Corynebacterium, Actinopolyspora,Nocardia, Saccharopolyspora, Nocardioides, or Granulicoccus. Thoughspecific examples are provided herein, other examples of microbialcytochrome P450 and reductase polypeptides are within the scope of thisdisclosure.

Cytochrome P450 polypeptides may be combined with reductase polypeptidesto form a functional two-component complex capable of dealkylating anaromatic substrate. One of both of the polypeptides may be used inpurified form. One of both of the polypeptides may be expressed by amicrobial biocatalyst to carry out dealkylation. A biocatalyst host cellmay express one or more of the polypeptides, or one or more of thepolypeptides may be added exogenously to a biocatalyst culture. Thecytochrome P450 and reductase polypeptides may be from the same organismor from different organisms, and various combinations may be created andtested for enzymatic activity.

TABLE 1 Cytochrome P450s and Reductases Reductases Cytochrome P450sOrganism Accession No. Organism Accession No. Rhodococcus pyridinivoransWP_006553157.1 Rhodococcus pyridinivorans WP_024102362.1 Rhodococcuspyridinivorans WP_041803486.1 Rhodococcus pyridinivorans WP_060652632.1Rhodococcus rhodochrous WP_016691158.1 Rhodococcus rhodochrousWP_016691159.1 Rhodococcus rhodochrous WP_059382679.1 Rhodococcus ruberWP_003934526.1 Rhodococcus ruber WP_003934527.1 Gordonia rhizospheraWP_006338923.1 Rhodococcus aetherivorans WP_029546517.1 Gordoniabronchialis WP_012835331.1 Rhodococcus wratislaviensis WP_037225711.1Gordonia namibiensis WP_006865278.1 Rhodococcus opacus WP_005261032.1Gordonia alkanivorans WP_006358554.1 Rhodococcus imtechensisWP_007297192.1 Gordonia rubripertincta WP_005194215.1 Rhodococcus jostiiWP_054246359.1 Gordonia terrae WP_004020850.1 Pimelobacter simplexWP_038682083.1 Rhodococcus wratislaviensis WP_037225709.1 Amycolatopsismethanolica WP_017982757.1 Rhodococcus jostii WP_011595125.1Amycolatopsis orientalis WP_043838556.1 Rhodococcus imtechensisWP_007297193.1 Amycolatopsis sp. ATCC 39116 WP_020419854.1 Rhodococcusopacus WP_012689299.1 Pseudonocardia autotrophica WP_037048581.1Amycolatopsis sp. ATCC39116 WP_020419855.1 Gordonia alkanivoransWP_006358553.1 Amycolatopsis methanolica WP_017982758.1 Gordonia sp.KTR9 WP_014924832.1 Gordonia polyisoprenivorans WP_006367698.1 Gordonianamibiensis WP_006865279.1 Gordonia polyisoprenivorans WP_014360659.1Gordonia terrae WP_004020851.1 Saccharomonospora cyanea WP_005457512.1Gordonia rubripertincta WP_039879854.1 Pseudonocardia autotrophicaWP_037048579.1 Saccharomonospora cyanea WP_005457513.1 Gordoniadesulfuricans WP_059037021.1 Gordonia polyisoprenivorans WP_014360660.1Nocardia farcinica WP_011208761.1 Gordonia bronchialis WP_012835332.1Saccharopolyspora rectivirgula WP_029722698.1 Gordonia rhizospheraWP_006338924.1 Nocardioides luteus WP_045548139.1 Gordoniarubripertincta GAB83512.1 Amycolatopsis orientalis WP_043838558.1Corynebacterium halotolerans WP_015400585.1 Amycolatopsis orientalisWP_037363061.1 Actinopolyspora halophila WP_017975556.1 Pseudonocardiasp. AL041005-10 ALE78654.1 Gordonia sputi WP_039856233.1 Granulicoccusphenolivorans WP_035757215.1 Rhodococcus wratislaviensis IFP 2016ELB87463.1 Pimelobacter simplex WP_038682080.1 Rhodococcus imtechensisWP_007296205.1 Gordonia amicalis WP_024500047.1 Gordonia desulfuricansWP_059037022.1

Also presented are microorganisms engineered to express the enzymesdisclosed herein and their use to biologically dealkylate aromaticsubstrates. Dealkylation may be carried out be culturing suchmicroorganisms with a material containing aromatic substrates (e.g.,guaiacol, guaethol or anisole) and allowing the microorganisms toenzymatically complete the conversion. Although the Examples presentedherein exemplify the use of the bacterium P. putida, any microorganismcapable of carrying out the dealkylation of the substrate through theaddition of enzymes disclosed herein may be suitable. Exemplarymicroorganisms include bacteria, such as those from the genusPseudomonas. Specific examples include strains of Pseudomonas putida,such as P. putida KT2440.

Aromatic substrate-containing materials may be contacted with enzymesdisclosed herein to dealkylate the substrate. As used herein, “aromaticsubstrate-containing materials” means any natural or processed materialscomprising detectable amounts of compounds such as guaiacol, guaethol oranisole. These may be derived from many sources, includinglignocellulose, lignin, or oils derived from the pyrolysis of biomass ofother lignocellulose or cellulose sources.

Suitable enzymes may be derived from microorganisms such as bacteria,fungi, yeast or the like via cell lysis and isolation techniques, orproduced recombinantly. In some embodiments, a microorganism expressingthe enzyme may be used directly as a biocatalyst to covert the aromaticsubstrate.

Enzymes described herein may be used as purified recombinant enzyme oras culture broths from cells that naturally produce the enzyme or thathave been engineered to produce the enzyme. Enzymes can be addedexogenously, or may be expressed and secreted directly from a microbialbiocatalyst, or used internally by the microbial biocatalyst. Suitableorganisms for enzyme expression include aerobic microorganisms such asaerobic bacteria.

Bio-oils and other aromatic substrate-containing materials may becontacted with enzymes at a concentration and a temperature for a timesufficient to achieve the desired amount of dealkylation. Suitable timesfor dealkylation range from a few hours to several days, and may beselected to achieve a desired amount of conversion. Exemplary reactiontimes include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours; and 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 days. In someembodiments, reaction times may be one or more weeks.

The resulting catechol, phenol, and the like may be further converted toproducts such as higher alcohols, hydrocarbons, or other advanced fuelsvia biological or chemical pathways, or combination thereof. Catechol,phenol and other products may be recovered or isolated from cells, cellcultures or reactions by standard separation techniques for furtherupgrading. Dealkylation products may also be further metabolized bybiocatalyst cells in the culture to additional products metabolicallyderived from a dealkylation product. These additional products may inturn be isolated from cells, cell cultures or reactions by standardseparation techniques and may be further upgraded to additional fuelsand chemicals.

Methods of fractionating, isolating or purifying dealkylation products(or further upgraded products) include a variety of biochemicalengineering unit operations. For example, the reaction mixture or cellculture lysate may be filtered to separate solids from products presentin a liquid portion. Dealkylation products may be further extracted froma solvent and/or purified using conventional methods. Exemplary methodsfor purification/isolation/separation of dealkylation products includeat least one of affinity chromatography, ion exchange chromatography,solvent extraction, filtration, centrifugation, electrophoresis,hydrophobic interaction chromatography, gel filtration chromatography,reverse phase chromatography, chromatofocusing, differentialsolubilization, preparative disc-gel electrophoresis, isoelectricfocusing, HPLC, and/or or reversed-phase HPLC.

Pyrolysis offers a straightforward approach for the deconstruction ofplant cell wall polymers into pyrolysis oil or bio-oil, which may befractionated and subsequently used in biological approaches toselectively upgrade some of the resulting fractions. Lignocellulose orlignin-containing materials may be subjected to pyrolysis processes togenerate oils containing aromatic substrates. Exemplarylignocellulose-containing materials include bioenergy crops,agricultural residues, municipal solid waste, industrial solid waste,sludge from paper manufacture, yard waste, wood and forestry waste.Examples of biomass include, but are not limited to, corn grain, corncobs, crop residues such as corn husks, corn stover, corn fiber,grasses, wheat, wheat straw, barley, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, wood(e.g., poplar) chips, sawdust, shrubs and bushes, vegetables, fruits,flowers and animal manure.

SEQ ID NOS: 1, 3, 5, 7, 9 and 11 provide nucleic acid and amino acidsequences for exemplary enzymes for use in the disclosed methods.“Nucleic acid” or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides.This includes single- and double-stranded molecules (i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

Nucleic acids referred to herein as “isolated” are nucleic acids thathave been removed from their natural milieu or separated away from thenucleic acids of the genomic DNA or cellular RNA of their source oforigin (e.g., as it exists in cells or in a mixture of nucleic acidssuch as a library), and may have undergone further processing. Isolatednucleic acids include nucleic acids obtained by methods describedherein, similar methods or other suitable methods, including essentiallypure nucleic acids, nucleic acids produced by chemical synthesis, bycombinations of biological and chemical methods, and recombinant nucleicacids that are isolated.

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures that rely upon a methodof artificial replication, such as the polymerase chain reaction (PCR)and/or cloning or assembling into a vector using restriction enzymes.Recombinant nucleic acids also include those that result fromrecombination events that occur through the natural mechanisms of cells,but are selected for after the introduction to the cells of nucleicacids designed to allow or make probable a desired recombination event.Portions of isolated nucleic acids that code for polypeptides having acertain function can be identified and isolated by, for example, themethod disclosed in U.S. Pat. No. 4,952,501.

An isolated nucleic acid molecule can be isolated from its naturalsource or produced using recombinant DNA technology (e.g., polymerasechain reaction (PCR) amplification, cloning or assembling) or chemicalsynthesis. Isolated nucleic acid molecules can include, for example,genes, natural allelic variants of genes, coding regions or portionsthereof, and coding and/or regulatory regions modified by nucleotideinsertions, deletions, substitutions, and/or inversions in a manner suchthat the modifications do not substantially interfere with the nucleicacid molecule's ability to encode a polypeptide or to form stablehybrids under stringent conditions with natural gene isolates. Anisolated nucleic acid molecule can include degeneracies. As used herein,nucleotide degeneracy refers to the phenomenon that one amino acid canbe encoded by different nucleotide codons. Thus, the nucleic acidsequence of a nucleic acid molecule that encodes a protein orpolypeptide can vary due to degeneracies.

Unless so specified, a nucleic acid molecule is not required to encode aprotein having enzyme activity. A nucleic acid molecule can encode atruncated, mutated or inactive protein, for example. In addition,nucleic acid molecules may also be useful as probes and primers for theidentification, isolation and/or purification of other nucleic acidmolecules, independent of a protein-encoding function.

Suitable nucleic acids include fragments or variants that encode afunctional enzyme. For example, a fragment can comprise the minimumnucleotides required to encode a functional cytochrome P450 O-dealkylaseor reductase. Nucleic acid variants include nucleic acids with one ormore nucleotide additions, deletions, substitutions, includingtransitions and transversions, insertion, or modifications (e.g., viaRNA or DNA analogs). Alterations may occur at the 5′ or 3′ terminalpositions of the reference nucleotide sequence or anywhere between thoseterminal positions, interspersed either individually among thenucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

In certain embodiments, a nucleic acid may be identical to a sequencerepresented herein. In other embodiments, the nucleic acids may be atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequencerepresented herein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to asequence represented herein. Sequence identity calculations can beperformed using computer programs, hybridization methods, orcalculations. Exemplary computer program methods to determine identityand similarity between two sequences include, but are not limited to,the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA. The BLASTprograms are publicly available from NCBI and other sources. Forexample, nucleotide sequence identity can be determined by comparingquery sequences to sequences in publicly available sequence databases(NCBI) using the BLASTN2 algorithm.

Embodiments of the nucleic acids include those that encode thepolypeptides that function as an O-dealkylase or a reductase orfunctional equivalents thereof. A functional equivalent includesfragments or variants of these that exhibit the ability to function asan O-dealkylase or a reductase. As a result of the degeneracy of thegenetic code, many nucleic acid sequences can encode a given polypeptidewith a particular enzymatic activity. Such functionally equivalentvariants are contemplated herein.

Nucleic acids may be derived from a variety of sources including DNA,cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Suchsequences may comprise genomic DNA, which may or may not includenaturally occurring introns. Moreover, such genomic DNA may be obtainedin association with promoter regions or poly (A) sequences. Thesequences, genomic DNA, or cDNA may be obtained in any of several ways.Genomic DNA can be extracted and purified from suitable cells by meanswell known in the art. Alternatively, mRNA can be isolated from a celland used to produce cDNA by reverse transcription or other means.

Also disclosed herein are recombinant vectors, including expressionvectors, containing nucleic acids encoding enzymes. A “recombinantvector” is a nucleic acid molecule that is used as a tool formanipulating a nucleic acid sequence of choice or for introducing such anucleic acid sequence into a host cell. A recombinant vector may besuitable for use in cloning, assembling, sequencing, or otherwisemanipulating the nucleic acid sequence of choice, such as by expressingor delivering the nucleic acid sequence of choice into a host cell toform a recombinant cell. Such a vector typically contains heterologousnucleic acid sequences not naturally found adjacent to a nucleic acidsequence of choice, although the vector can also contain regulatorynucleic acid sequences (e.g., promoters, untranslated regions) that arenaturally found adjacent to the nucleic acid sequences of choice or thatare useful for expression of the nucleic acid molecules.

The nucleic acids described herein may be used in methods for productionof enzymes and enzyme cocktails through incorporation into cells,tissues, or organisms. In some embodiments, a nucleic acid may beincorporated into a vector for expression in suitable host cells. Thevector may then be introduced into one or more host cells by any methodknown in the art. One method to produce an encoded protein includestransforming a host cell with one or more recombinant nucleic acids(such as expression vectors) to form a recombinant cell. The term“transformation” is generally used herein to refer to any method bywhich an exogenous nucleic acid molecule (i.e., a recombinant nucleicacid molecule) can be inserted into a cell, but can be usedinterchangeably with the term “transfection.”

Non-limiting examples of suitable host cells include cells frommicroorganisms such as bacteria, yeast, fungi, and filamentous fungi.Exemplary microorganisms include, but are not limited to, bacteria suchas E. coli; bacteria from the genera Pseudomonas (e.g., P. putida or P.fluorescens), Bacillus (e.g., B. subtilis, B. megaterium or B. brevis),Caulobacter (e.g., C. crescentus), Lactoccocus (e.g., L. lactis),Streptomyces (e.g., S. coelicolor), Streptococcus (e.g., S. lividans),and Corynybacterium (e.g., C. glutamicum); fungi from the generaTrichoderma (e.g., T. reesei, T. viride, T. koningii, or T. harzianum),Penicillium (e.g., P. funiculosum), Humicola (e.g., H. insolens),Chrysosporium (e.g., C. lucknowense), Gliocladium, Aspergillus (e.g., A.niger, A. nidulans, A. awamori, or A. aculeatus), Fusarium, Neurospora,Hypocrea (e.g., H. jecorina), and Emericella; yeasts from the generaSaccharomyces (e.g., S. cerevisiae), Pichia (e.g., P. pastoris), orKluyveromyces (e.g., K. lactis). Cells from plants such as Arabidopsis,barley, citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat,switch grass, alfalfa, miscanthus, and trees such as hardwoods andsoftwoods are also contemplated herein as host cells.

Host cells can be transformed, transfected, or infected as appropriateby any suitable method including electroporation, calcium chloride-,lithium chloride-, lithium acetate/polyene glycol-, calcium phosphate-,DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection,microinjection, microprojectile bombardment, phage infection, viralinfection, or other established methods. Alternatively, vectorscontaining the nucleic acids of interest can be transcribed in vitro,and the resulting RNA introduced into the host cell by well-knownmethods, for example, by injection. Exemplary embodiments include a hostcell or population of cells expressing one or more nucleic acidmolecules or expression vectors described herein (for example, agenetically modified microorganism). The cells into which nucleic acidshave been introduced as described above also include the progeny of suchcells.

Vectors may be introduced into host cells such as those from bacteria orfungi by direct transformation, in which DNA is mixed with the cells andtaken up without any additional manipulation, by conjugation,electroporation, or other means known in the art. Expression vectors maybe expressed by bacteria or fungi or other host cells episomally or thegene of interest may be inserted into the chromosome of the host cell toproduce cells that stably express the gene with or without the need forselective pressure. For example, expression cassettes may be targeted toneutral chromosomal sites by recombination.

Host cells carrying an expression vector (i.e., transformants or clones)may be selected using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule.In prokaryotic hosts, the transformant may be selected, for example, byresistance to ampicillin, tetracycline or other antibiotics. Productionof a particular product based on temperature sensitivity may also serveas an appropriate marker.

Host cells may be cultured in an appropriate fermentation medium. Anappropriate, or effective, fermentation medium refers to any medium inwhich a host cell, including a genetically modified microorganism, whencultured, is capable of growing or expressing the polypeptides describedherein. Such a medium is typically an aqueous medium comprisingassimilable carbon, nitrogen and phosphate sources, but can also includeappropriate salts, minerals, metals and other nutrients. Microorganismsand other cells can be cultured in conventional fermentation bioreactorsand by any fermentation process, including batch, fed-batch, cellrecycle, and continuous fermentation. The pH of the fermentation mediumis regulated to a pH suitable for growth of the particular organism.Culture media and conditions for various host cells are known in theart. A wide range of media for culturing bacteria or fungi, for example,are available from ATCC. Exemplary culture/fermentation conditions andreagents are provided in the Examples that follow. Media may besupplemented with aromatic substrates like guaiacol, guaethol or anisolefor dealkylation reactions.

The nucleic acid molecules described herein encode the enzymes withamino acid sequences such as those represented by SEQ ID NOS:2, 4, 6, 8,10 and 12. As used herein, the terms “protein” and “polypeptide” aresynonymous. “Peptides” are defined as fragments or portions ofpolypeptides, preferably fragments or portions having at least onefunctional activity as the complete polypeptide sequence. “Isolated”proteins or polypeptides are proteins or polypeptides purified to astate beyond that in which they exist in cells. In certain embodiments,they may be at least 10% pure; in others, they may be substantiallypurified to 80% or 90% purity or greater. Isolated proteins orpolypeptides include essentially pure proteins or polypeptides, proteinsor polypeptides produced by chemical synthesis or by combinations ofbiological and chemical methods, and recombinant proteins orpolypeptides that are isolated. Proteins or polypeptides referred toherein as “recombinant” are proteins or polypeptides produced by theexpression of recombinant nucleic acids.

Proteins or polypeptides encoded by nucleic acids as well as functionalportions or variants thereof are also described herein. Polypeptidesequences may be identical to the amino acid sequences presented in SEQID NOS:2, 4, 6, 8, 10 and 12, or may include up to a certain integernumber of amino acid alterations. Such protein or polypeptide variantsretain functionality as a cytochrome P450 O-dealkylase or a reductase,and include mutants differing by the addition, deletion or substitutionof one or more amino acid residues, or modified polypeptides and mutantscomprising one or more modified residues. The variant may have one ormore conservative changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). Alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence.

In certain embodiments, the polypeptides may be at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequences set forth inSEQ ID NOS:2, 4, 6, 8, 10 and 12 and possess enzymatic function. Percentsequence identity can be calculated using computer programs (such as theBLASTP and TBLASTN programs publicly available from NCBI and othersources) or direct sequence comparison. Polypeptide variants can beproduced using techniques known in the art including directmodifications to isolated polypeptides, direct synthesis, ormodifications to the nucleic acid sequence encoding the polypeptideusing, for example, recombinant DNA techniques.

Polypeptides may be retrieved, obtained, or used in “substantially pure”form, a purity that allows for the effective use of the protein in anymethod described herein or known in the art. For a protein to be mostuseful in any of the methods described herein or in any method utilizingenzymes of the types described herein, it is most often substantiallyfree of contaminants, other proteins and/or chemicals that mightinterfere or that would interfere with its use in the method (e.g., thatmight interfere with enzyme activity), or that at least would beundesirable for inclusion with a protein.

EXAMPLES Example 1 Enzyme Cloning

The primers listed in Table 2 were used in the gene amplificationsdescribed below.

TABLE 2 Nucleic Acid Sequences of Primers Primer Sequence (5′-3′)SEQ ID NO. oCJ160 GATATCATTCAGGACGAGCCTCAGACTCC SEQ ID NO: 13 oCJ161CTCTAGAGTGTGAAATTGTTATCCGCTCA SEQ ID NO: 14 CAATTCC oCJ169AACAATTTCACACTCTAGAGAGGAGGACA SEQ ID NO: 15 GCTATGACGACGACCGAACGGCCoCJ170 GGCTCGTCCTGAATGATATCTCACGAGGC SEQ ID NO: 16 CGGCGTG oCJ206GGCTCGTCCTGAATGATATCTCACACCTC SEQ ID NO: 17 CCAGGTGACGTG

The genes encoding the cytochrome P450 protein and the co-transcribedreductase were amplified from Amycolatopsis sp. ATCC 39116 DNA usingprimers oCJ169 and oCJ170 (see Table 2) and the product assembled usingGibson Assembly Master Mix (New England BioLabs) into the broadhost-range vector pBTL-2, which was amplified using primers oCJ160 andoCJ161, resulting in pCJ021. pBTL-2 (a gift from Ryan Gill; Addgeneplasmid #22806) is a broad host-range vector that drives expression ofinserted genes using the lac promoter, which is constitutively expressedin Pseudomonas putida KT2440. The cytochrome P450 gene alone was alsoamplified using oCJ169 and oCJ206 and assembled it into the same vector(resulting in pCJ024). pCJ021 was then transformed into P. putida KT2440to generate P. putida KT2440/pCJ021.

Example 2 HPLC Analyses

HPLC analysis was performed by injecting 6 μL of 0.2 μm filtered culturesupernatant onto an Agilent 1100 series system equipped with aPhenomenex Rezex RFQ-Fast Acid H+ (8%) column and a cation H+ guardcartridge (Bio-Rad Laboratories) at 85° C. run using a mobile phase of0.01 N sulfuric acid at a flow rate of 1.0 mL/min and a diode arraydetector to measure absorbance at 210 nm. Analytes were identified bycomparing retention times and spectral profiles with pure standards.HPLC chromatograms show milli-absorbance units (mAU) on the Y axis andretention time (minutes) on the X axis.

Example 3 Conversion of Guaiacol, Anisole and Guaethol

P. putida KT2440 can readily metabolize catechol for growth, so afunctional enzyme with O-dealkylase activity would convert guaiacol orguaethol to catechol and enable this organism to metabolize guaiacol orguaethol for growth, which it is not natively capable of P. putidaKT2440 transformed with pCJ021 (KT2440/pCJ021) was capable of growth inM9 minimal medium containing 5 mM guaiacol as the sole source of carbonand energy, metabolizing the guaiacol completely and reaching an OD₆₀₀of just over 0.5 (FIG. 4). Native P. putida KT2440 and P. putida KT2440transformed with pCJ024 were included in this experiment, but neither ofthese strains was able to metabolize guaiacol.

HPLC analyses show the disappearance of guaiacol in a culture of P.putida KT2440/pCJ021 grown on M9 minimal medium containing guaiacol andthe lack of guaiacol catabolism by native P. putida KT2440 (FIG. 6).When these strains are grown in LB medium containing guaiacol, HPLCanalysis shows the conversion of guaiacol to catechol (an increase incatechol levels and a corresponding decrease in guaiacol levels), whichtemporarily accumulated during catabolism of guaiacol (FIG. 5). Theseresults indicate that the amplified genes encode a two-componentcytochrome P450 system with guaiacol O-demethylase activity.

Conversion of anisole was demonstrated by growing P. putidaKT2440/pCJ021 in LB medium containing anisole. The cultures were grownin LB, a rich medium, because P. putida KT2440 cannot metabolize phenol.HPLC analysis of cultures of P. putida KT2440/pCJ021 grown in LB mediumcontaining anisole demonstrated this strain catalyzed the conversion ofanisole to phenol, which accumulates because P. putida KT2440 cannotmetabolize it further. Control cultures of P. putida KT2440 did notaccumulate phenol (FIG. 7). A peak corresponding to anisole was notobserved due to the HPLC column used. The production of phenol, however,demonstrates that anisole is being demethylated to generate phenol in P.putida KT2440/pCJ021 cultures.

Dealkylation of guaethol was demonstrated by growing P. putidaKT2440/pCJ021 in M9 minimal medium containing 5 mM guaethol(2-ethoxyphenol). After 72 hours of growth with guaethol as the solesource of carbon and energy, P. putida KT2440/pCJ021 was able tometabolize the guaethol in the culture while native P. putida KT2440 wasunable to metabolize guaethol. HPLC analysis shows the disappearance ofguaethol in a culture of P. putida KT2440/pCJ021 after 72 hours, but notin a culture of native P. putida KT2440 (FIG. 8). P. putidaKT2440/pCJ021 was able to grow to an OD₆₀₀ of 7 on the catechol producedin this reaction, as demonstrated in FIG. 9.

Taken together, these data demonstrate that the two-component cytochromeP450 system catalyzes the O-demethylation of guaiacol (2-methoxyphenol)to catechol (1,2-dihydroxybenzene), the O-demethylation of anisole(methoxybenzene) to phenol, and the O-deethylation of guaethol(2-ethoxyphenol) to catechol (FIG. 1).

The Examples discussed above are provided for purposes of illustrationand are not intended to be limiting. Still other embodiments andmodifications are also contemplated.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

We claim:
 1. A method for removing an alkyl group from an aromaticsubstrate, comprising contacting a material containing the aromaticsubstrate with a cytochrome P450 polypeptide and a reductase polypeptideto generate a dealkylation product.
 2. The method of claim 1, whereinthe contacting step comprises culturing a microorganism with thematerial containing the aromatic substrate, wherein the microorganismexpresses an exogenous gene encoding a cytochrome P450 polypeptide or areductase polypeptide.
 3. The method of claim 1, wherein the aromaticsubstrate comprises guaiacol, anisole or guaethol.
 4. The method ofclaim 1, wherein the material containing the aromatic substratecomprises products of lignin depolymerization.
 5. The method of claim 1,wherein the material containing the aromatic substrate comprises apyrolysis oil or bio-oil.
 6. The method of claim 1, wherein at least oneof the cytochrome P450 polypeptide or the reductase polypeptide is froma bacterium.
 7. The method of claim 1, wherein at least one of thecytochrome P450 polypeptide or the reductase polypeptide is from abacterium from the genera Amycolatopsis or Rhodococcus.
 8. The method ofclaim 1, wherein the cytochrome P450 polypeptide has an amino acidsequence at least 90% identical to SEQ ID NO:2.
 9. The method of claim1, wherein the reductase polypeptide has an amino acid sequence at least90% identical to SEQ ID NO:4.
 10. The method of claim 1, wherein thedealkylation product is catechol or phenol.
 11. The method of claim 1,further comprising isolating the dealkylation product.
 12. An isolatedcDNA molecule encoding a cytochrome P450 polypeptide that has an aminoacid sequence at least 90% identical to SEQ ID NO:2 or encoding areductase polypeptide that has an amino acid sequence at least 90%identical to SEQ ID NO:4.
 13. An isolated cDNA molecule of claim 12,wherein the polypeptide has the amino acid sequence of SEQ ID NO:2. 14.An isolated cDNA molecule of claim 12, wherein the polypeptide has theamino acid sequence of SEQ ID NO:4.
 15. The isolated cDNA molecule ofclaim 1, further comprising an exogenous promoter operably linked to thecDNA molecule.
 16. An expression vector comprising the cDNA molecule ofclaim
 12. 17. A host cell that expresses a recombinant polypeptideencoded by the cDNA molecule of claim
 12. 18. The host cell of claim 17,wherein the cell is from a strain of Pseudomonas.
 19. The host cell ofclaim 18, wherein the cell is P. putida.
 20. An isolated cytochrome P450polypeptide or a reductase polypeptide encoded by the cDNA molecule ofclaim 12.